Central autonomic control in spontaneously hypertensive rats: a study on phasic phenomena during rapid-eye-movement sleep

The cardiovascular regulation undergoes wide changes in the different states of sleepwake cycle. In particular, the relationship between spontaneous fluctuations in heart period and arterial pressure clearly shows differences between the two sleep states. In non rapid-eye-movement sleep, heart rhythm is under prevalent baroreflex control, whereas in rapid-eye-movement sleep central autonomic commands prevail (Zoccoli et al., 2001). Moreover, during rapid-eye-movement sleep the cardiovascular variables show wide fluctuations around their mean value. In particular, during rapid-eyemovement sleep, the arterial pressure shows phasic hypertensive events which are superimposed upon the tonic level of arterial pressure. These phasic increases in arterial pressure are accompanied by an increase in heart rate (Sei & Morita, 1996; Silvani et al., 2005). Thus, rapid-eye-movement sleep may represent an “autonomic stress test” for the cardiovascular system, able to unmask pathological patterns of cardiovascular regulation (Verrier et al. 2005), but this hypothesis has never been tested experimentally. The aim of this study was to investigate whether rapid-eye-movement sleep may reveal derangements in central autonomic cardiovascular control in an experimental model of essential hypertension. The study was performed in Spontaneously Hypertensive Rats, which represent the most widely used model of essential hypertension, and allow full control of genetic and environmental confounding factors. In particular, we analyzed the cardiovascular, electroencephalogram, and electromyogram changes associated with phasic hypertensive events during rapid-eyemovement sleep in Spontaneously Hypertensive Rats and in their genetic Wistar Kyoto control strain. Moreover, we studied also a group of Spontaneously Hypertensive Rats made phenotypically normotensive by means of a chronic treatment with an angiotensin converting enzyme inhibitor, the Enalapril maleate, from the age of four weeks to the end of the experiment. All rats were implanted with electrodes for electroencephalographic and electromyographic recordings and with an arterial catheter for arterial pressure measurement. After six days for postoperative recovery, the rats were studied for five days, at an age of ten weeks.The study indicated that the peak of mean arterial pressure increase during the phasic hypertensive events in rapid-eye-movement sleep did not differ significantly between Spontaneously Hypertensive Rats and Wistar Kyoto rats, while on the other hand Spontaneously Hypertensive Rats showed a reduced increase in the frequency of theta rhythm and a reduced tachicardia with respect to Wistar Kyoto rats. The same pattern of changes in mean arterial pressure, heart period, and theta frequency was observed between Spontaneously Hypertensive Rats and Spontaneously Hypertensive Rats treated with Enalapril maleate. Spontaneously Hypertensive Rats do not differ from Wistar Kyoto rats only in terms of arterial hypertension, but also due to multiple unknown genetic differences. Spontaneously Hypertensive Rats were developed by selective breeding of Wistar Kyoto rats based only on the level of arterial pressure. However, in this process, multiple genes possibly unrelated to hypertension may have been selected together with the genetic determinants of hypertension (Carley et al., 2000). This study indicated that Spontaneously Hypertensive Rats differ from Wistar Kyoto rats, but not from Spontaneously Hypertensive Rats treated with Enalapril maleate, in terms of arterial pH and theta frequency. This feature may be due to genetic determinants unrelated to hypertension. In sharp contrast, the persistence of differences in the peak of heart period decrease and the peak of theta frequency increase during phasic hypertensive events between Spontaneously Hypertensive Rats and Spontaneously Hypertensive Rats treated with Enalapril maleate demonstrates that the observed reduction in central autonomic control of the cardiovascular system in Spontaneously Hypertensive Rats is not an irreversible consequence of inherited genetic determinants. Rather, the comparison between Spontaneously Hypertensive Rats and Spontaneously Hypertensive Rats treated with Enalapril maleate indicates that the observed differences in central autonomic control are the result of the hypertension per se. This work supports the view that the study of cardiovascular regulation in sleep provides fundamental insight on the pathophysiology of hypertension, and may thus contribute to the understanding of this disease, which is a major health problem in European countries (Wolf-Maier et al., 2003) with its burden of cardiac, vascular, and renal complications.

Identification of Drosophila heart-specific Cis-Regulatory Modules under Hox control

Cardiac morphogenesis is a complex process governed by evolutionarily conserved transcription factors and signaling molecules. The Drosophila cardiac tube is linear, made of 52 pairs of cardiomyocytes (CMs), which express specific transcription factor genes that have human homologues implicated in Congenital Heart Diseases (CHDs) (NKX2-5, GATA4 and TBX5). The Drosophila cardiac tube is linear and composed of a rostral portion named aorta and a caudal one called heart, distinguished by morphological and functional differences controlled by Hox genes, key regulators of axial patterning. Overexpression and inactivation of the Hox gene abdominal-A (abd-A), which is expressed exclusively in the heart, revealed that abd-A controls heart identity. The aim of our work is to isolate the heart-specific cisregulatory sequences of abd-A direct target genes, the realizator genes granting heart identity. In each segment of the heart, four pairs of cardiomyocytes (CMs) express tinman (tin), homologous to NKX2-5, and acquire strong contractile and automatic rhythmic activities. By tyramide amplified FISH, we found that seven genes, encoding ion channels, pumps or transporters, are specifically expressed in the Tin-CMs of the heart. We initially used online available tools to identify their heart-specific cisregutatory modules by looking for Conserved Non-coding Sequences containing clusters of binding sites for various cardiac transcription factors, including Hox proteins. Based on these data we generated several reporter gene constructs and transgenic embryos, but none of them showed reporter gene expression in the heart. In order to identify additional abd-A target genes, we performed microarray experiments comparing the transcriptomes of aorta versus heart and identified 144 genes overexpressed in the heart. In order to find the heart-specific cis-regulatory regions of these target genes we developed a new bioinformatic approach where prediction is based on pattern matching and ordered statistics. We first retrieved Conserved Noncoding Sequences from the alignment between the D.melanogaster and D.pseudobscura genomes. We scored for combinations of conserved occurrences of ABD-A, ABD-B, TIN, PNR, dMEF2, MADS box, T-box and E-box sites and we ranked these results based on two independent strategies. On one hand we ranked the putative cis-regulatory sequences according to best scored ABD-A biding sites, on the other hand we scored according to conservation of binding sites. We integrated and ranked again the two lists obtained independently to produce a final rank. We generated nGFP reporter construct flies for in vivo validation. We identified three 1kblong heart-specific enhancers. By in vivo and in vitro experiments we are determining whether they are direct abd-A targets, demonstrating the role of a Hox gene in the realization of heart identity. The identified abd-A direct target genes may be targets also of the NKX2-5, GATA4 and/or TBX5 homologues tin, pannier and Doc genes, respectively. The identification of sequences coregulated by a Hox protein and the homologues of transcription factors causing CHDs, will provide a mean to test whether these factors function as Hox cofactors granting cardiac specificity to Hox proteins, increasing our knowledge on the molecular mechanisms underlying CHDs. Finally, it may be investigated whether these Hox targets are involved in CHDs.

Maximum principle, mean value operators and quasi boundedness in non-euclidean settings

This work deals with some classes of linear second order partial differential operators with non-negative characteristic form and underlying non- Euclidean structures. These structures are determined by families of locally Lipschitz-continuous vector fields in RN, generating metric spaces of Carnot- Carath´eodory type. The Carnot-Carath´eodory metric related to a family {Xj}j=1,...,m is the control distance obtained by minimizing the time needed to go from two points along piecewise trajectories of vector fields. We are mainly interested in the causes in which a Sobolev-type inequality holds with respect to the X-gradient, and/or the X-control distance is Doubling with respect to the Lebesgue measure in RN. This study is divided into three parts (each corresponding to a chapter), and the subject of each one is a class of operators that includes the class of the subsequent one. In the first chapter, after recalling “X-ellipticity” and related concepts introduced by Kogoj and Lanconelli in [KL00], we show a Maximum Principle for linear second order differential operators for which we only assume a Sobolev-type inequality together with a lower terms summability. Adding some crucial hypotheses on measure and on vector fields (Doubling property and Poincar´e inequality), we will be able to obtain some Liouville-type results. This chapter is based on the paper [GL03] by Guti´errez and Lanconelli. In the second chapter we treat some ultraparabolic equations on Lie groups. In this case RN is the support of a Lie group, and moreover we require that vector fields satisfy left invariance. After recalling some results of Cinti [Cin07] about this class of operators and associated potential theory, we prove a scalar convexity for mean-value operators of L-subharmonic functions, where L is our differential operator. In the third chapter we prove a necessary and sufficient condition of regularity, for boundary points, for Dirichlet problem on an open subset of RN related to sub-Laplacian. On a Carnot group we give the essential background for this type of operator, and introduce the notion of “quasi-boundedness”. Then we show the strict relationship between this notion, the fundamental solution of the given operator, and the regularity of the boundary points.

NLGA based Active Control in Aerospace: fault tolerability, disturbance rejection, and parameter estimation

A new control scheme has been presented in this thesis. Based on the NonLinear Geometric Approach, the proposed Active Control System represents a new way to see the reconfigurable controllers for aerospace applications. The presence of the Diagnosis module (providing the estimation of generic signals which, based on the case, can be faults, disturbances or system parameters), mean feature of the depicted Active Control System, is a characteristic shared by three well known control systems: the Active Fault Tolerant Controls, the Indirect Adaptive Controls and the Active Disturbance Rejection Controls. The standard NonLinear Geometric Approach (NLGA) has been accurately investigated and than improved to extend its applicability to more complex models. The standard NLGA procedure has been modified to take account of feasible and estimable sets of unknown signals. Furthermore the application of the Singular Perturbations approximation has led to the solution of Detection and Isolation problems in scenarios too complex to be solved by the standard NLGA. Also the estimation process has been improved, where multiple redundant measuremtent are available, by the introduction of a new algorithm, here called "Least Squares - Sliding Mode". It guarantees optimality, in the sense of the least squares, and finite estimation time, in the sense of the sliding mode. The Active Control System concept has been formalized in two controller: a nonlinear backstepping controller and a nonlinear composite controller. Particularly interesting is the integration, in the controller design, of the estimations coming from the Diagnosis module. Stability proofs are provided for both the control schemes. Finally, different applications in aerospace have been provided to show the applicability and the effectiveness of the proposed NLGA-based Active Control System.